Cell-free nucleic acids (CNAs) consist of extra-cellular genetic material freely found in human body fluids, including circulating body fluids. These information rich molecules are an excellent source of non-invasive biomarkers as they are readily released into the body from both pathologic and healthy cells. They are being extensively studied in a diverse array of human diseases such as cancer, fetal medicine, and diabetes (Swamp et al. (2007) FEBS Lett 581, 795-799; Tong et al. (2006) Clin Chim Acta 363, 187-196). In cancer, CNAs can be used to determine the status of remote tumors through the analysis of both genetic and epigenetic information, potentially bypassing the need for tissue biopsies. They have been demonstrated throughout cancer management in the assessment of tumor dynamics, therapeutic response, disease progression, and prognosis (Diehl et al. (2008) Nat Med 14, 985-990; Umetani et al. (2006) J Clin Oncol 24, 4270-4276; Wang et al. (2003) Cancer Res 63, 3966-3968).
Despite the potential benefits, the clinical analysis of CNA biomarkers faces two key hurdles. First, CNAs are present at very low concentrations within the body (˜10-400 ng/mL in plasma) and, second, CNAs of interest must be accurately discerned from a sea of obscuring background CNAs. To date, PCR-based methods have been used near exclusively due to its high sensitivity. While PCR has been very successful in biomarker discovery and research, it has intrinsic limitations. For example, PCR is a complicated enzymatic process that requires substantial optimization to obtain favorable results, making PCR finicky and somewhat difficult to reliably reproduce on a daily basis. More complicated variants, such as quantitative real-time PCR (qPCR) and nested PCR, require yet more care, making robust quantification and multiplex detection difficult. qPCR has wide dynamic range but is typically limited to measuring 2-fold changes in quantity. Multiplexed PCR reactions can also be affected by varying amplification efficiencies, leading to poor quantification accuracy. Furthermore, the sensitive nature of the PCR process and the lack of standardization in sample preparation steps can further exacerbate these issues. Practically, long run times, tedious sample processing steps, expensive reagents, and trained technicians required for PCR, limit its clinical utility.
One way of analyzing CNAs to detect cancer is the DNA Integrity Assay (DIA). This is a unique cancer biomarker that is highly specific to cancer in general, but not to cancer type. Rather than relying on sequence or epigenetic information contained within the CNAs, DIA uses the size distribution of CNA fragments to determine their origin. Apoptotic cells are postulated to release uniform pieces of 180 bp long DNA while tumor cells, which tend to die haphazardly through necrosis and cell lysis, release fragments of much longer and more variable length (Jahr et al. (2001) Cancer Res 61, 1659-1665). The apoptotic origins of typical short DNA strands have recently been verified by sequencing studies. DIA may have high utility in early detection and screening of cancer, particularly for cancers in which no widely accepted markers currently exist. It was first demonstrated for the detection of colon and gynecological cancers (Wang et al. (2003) Cancer Res 63, 3966-3968; Boynton et al. (2003) Clin Chem 49, 1058-1065) and subsequently for the detection of breast, nasopharyngeal, and prostate cancer (Umetani et al. (2006) J Clin Oncol 24, 4270-4276; Chan et al. (2008) Clin Cancer Res 14, 4141-4145; Hanley et al. (2006) Clin Cancer Res 12, 4569-4574). The size distribution of these cell-free DNAs may be used as a marker for cancer detection and potentially to monitor therapeutic efficacy and disease recurrence (Umetani et al. (2006, supra); Chan et al. (2008, supra).
There is a need to develop methods that can be used instead of, or in addition to, PCR to analyze CNAs and, in particular, to conduct DNA Integrity Assays (DIA).